XTT assay for detection of bacterial metabolic activity in water-based polyester polyurethane

Cellular metabolic activity can be detected by tetrazolium-based colorimetric assays, which rely on dehydrogenase enzymes from living cells to reduce tetrazolium compounds into colored formazan products. Although these methods have been used in different fields of microbiology, their application to the detection of bacteria with plastic-degrading activity has not been well documented. Here, we report a microplate-adapted method for the detection of bacteria metabolically active on the commercial polyester polyurethane (PU) Impranil®DLN using the tetrazolium salt 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT). Bacterial cells that are active on PU reduce XTT to a water-soluble orange dye, which can be quantitatively measured using a microplate reader. We used the Pseudomonas putida KT2440 strain as a study model. Its metabolic activity on Impranil detected by our novel method was further verified by Fourier-transform infrared spectroscopy (FTIR) analyses. Measurements of the absorbance of reduced XTT at 470 nm in microplate wells were not affected by the colloidal properties of Impranil or cell density. In summary, we provide here an easy and high-throughput method for screening bacteria active on PU that can be adapted to other plastic substrates.


Practical considerations
The assay can be adapted for other bacterial strains.Also, you can use an automated microplate spectrophotometer to directly incubate and measure the transformation of XTT to formazan continuously (i.e., every hour).

SAFETY WARNINGS
This procedure involves the use of hazardous chemicals (chloroform and methanol) for the glassware preparation.
XTT contains no substances with occupational exposure limit values.
1. Read the corresponding safety data sheets for each chemical in the procedure.
2. Use personal protective equipment throughout the procedure: nitrile gloves, wear a lab coat and safety glasses.
3. Dispose of all chemical waste in appropriately labeled containers.

Washing glassware
All glassware should be washed twice using 2 mL of methanol-chloroform mixture (1:1) and allowed to dry in a fume hood. 1 Scrape some of the frozen surface of the glycerol stock using a sterile loop and streak the bacteria onto a Luria-Bertani (LB) agar plate.

Note
Use adecuate incubation temperature for your strain.

3
Take one isolated colony and inoculate it into 5 mL LB broth. 4 Incubate Overnight at 30 °C with shaking at 180 rpm .

4.1
Measure the optical density (OD) at 600 nm of cultures using a spectrophotometer.

Note
If needed, prepare a dilution of the culture (e.g., 10 -1 ) using fresh LB broth and measure its OD 600 .The OD 600 of the original culture is calculated by multiplying the obtained OD by the dilution factor.

Note
Our research group uses marine salts to standardize the protocol and screen marine bacteria for subsequent toxicity assays of bacterial culture in zebra sh embryos.For your speci c requirements, feel free to use an appropriate minimal medium and carbon source.

Note
This 4 hour timeframe applies to our experimental and cultural conditions.It is important to be knowledgeable of the exponential growth rate of your bacterial strains.

7.1
Wash twice the cellular pellet by suspension in 20 mL of sterile 10mM MgSO 4 followed by centrifugation at 6000 rpm, 4°C, 00:10:00 to remove all traces of the old culture medium.

7.2
Resuspend the cells in 5 mL of fresh sterile 10mM MgSO 4 and reserve to be used as inoculum for the next steps.Keep the washed cells on ice to facilitate their handling and preparation.